This proposal will investigate properties of a class of molecules that transduce pain caused by ischemia (insufficient delivery of oxygen to an organ) and that might contribute to neuronal damage during stroke and seizure. The molecules are called acid-sensing ion channels (ASICs). They selectively pass sodium ions and calcium ions into cells when they are opened by a drop in extracellular pH. Such pH changes occur during ischemia when lactic acid is produced and they occur when there is hyperactivity in the CNS or when CNS blood vessels get occluded. The long-term objective is to determine whether ASlCs are appropriate pharmaceutical targets for: a) diminishing ischemic pain, and b) neuroprotection after stroke and seizure. Ischemia is the primary source of cardiac pain, sickle cell anemia pain, and the muscle pain of intermittent claudication; it may also contribute to other forms of muscle, bone, and visceral pain. [unreadable] The immediate goal is to describe the properties of ASlCs that seem critical to these pathological conditions. The specific aims arise from our preliminary results that demonstrate that ASlCs open through an unexpected mechanism. ASICs were thought to open because protons (lowered pH) trigger a conformation change in the protein. Instead, we find that protons open ASlC3 because they catalyze the release of a bound calcium ion that blocks the pore. This mechanism is important in disease because pH and calcium drop simultaneously in both peripheral and cerebral ischemia, providing two stimuli acting in concert to open ASlCs. [unreadable] The specific aims are: 1) to define the amino acids in ASlC3 that form the calcium/proton binding site that controls channel opening; 2) to define how ASlC1a, the dominant ASlC in the CNS, behaves under ionic conditions that occur during stroke and seizure; 3) to understand the basis of persistent currents through ASlC3 that occur around pH 7.0, the crucial range for ischemic muscle pain. Experiments utilize patch clamp electrophysiology and molecular mutagenesis of cloned ion channels [unreadable] [unreadable]